The long-term goal of this research is to determine the role(s) of Stat5 transcription factors in insulin signaling, specifically insulin- regulated gene transcription. Preliminary data suggest that activation of the insulin receptor (IR) in vivo leads to the specific tyrosine phosphorylation and activation of two members of the STAT family of transcription factors, Stat5a and Stat5b, in insulin target tissues of mice. It is hypothesized that Stat 5a and Stat5b are physiological mediators of insulin receptor signaling and contribute to the transcriptional regulation of some genes by insulin. Preliminary data also show that the IR can associate with Stat5b in yeast and directly phosphorylate this protein in vitro, raising the possibility that Stat5b, and perhaps Stat5a, represent physiological substrates of the IR tyrosine kinase. Aim 1 proposes to demonstrate that insulin directly activates Stat5 proteins in vivo and then characterize this response with regard to responding cell types, JAK activation and effects of counter-regulatory hormones. Diabetic, hypophysectomized and fasted- refed mice will be used, and selected insulin target tissues will be analyzed for Stat5 protein and JAK kinase activation. Aim 2 proposes to determine the molecular mechanisms(s) through which the IR mediates Stat5 protein activation. This goal will determine in cultured cells and in vitro whether the IR can phosphorylate Stat5 proteins directly or whether insulin-activated JAKs perform this function. Potential involvement of IRS-1 will also be determined. Aim 3 proposes to determine the role of Stat5 proteins in insulin-regulated gene transcription in muscle. This goal will be achieved through the stable expression of Stat5 dominant negative (DN) proteins in a differentiating skeletal muscle cell line to create Stat5 minus cells.